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Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin‐fixed paraffin‐embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale‐up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non‐TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics. [ABSTRACT FROM AUTHOR]

Tuberculosis (TB) is a global health problem. The immunohistochemistry (IHC)-based MPT64 antigen detection test has shown promising results for diagnosing extrapulmonary TB in previous studies. However, the anti-MPT64 antibody currently used in the test is in limited supply, and reproduction of a functional antibody is a prerequisite for further large-scale use. Various antigen-adjuvant combinations and immunisation protocols were tested in mice and rabbits to generate monoclonal and polyclonal antibodies. Antibodies were screened in IHC, and the final new antibody was validated on clinical human specimens. We were not able to generate monoclonal antibodies that were functional in IHC, but we obtained multiple functional polyclonal antibodies through careful selection of antigen-adjuvant and comprehensive screening in IHC of both pre-immune sera and antisera. To overcome the limitation of batch-to-batch variability with polyclonal antibodies, the best performing individual polyclonal antibodies were pooled to one final large-volume new anti-MPT64 antibody. The sensitivity of the new antibody was in the same range as the reference antibody, while the specificity was somewhat reduced. Our results suggest that it possible to reproduce a large-volume functional polyclonal antibody with stable performance, thereby securing stable supplies and reproducibility of the MPT64 test, albeit further validation remains to be done. [ABSTRACT FROM AUTHOR]